Department of Life Science, National Taiwan Ocean University, Keelung, Taiwan.
Appl Biochem Biotechnol. 2011 Oct;165(3-4):1047-56. doi: 10.1007/s12010-011-9319-7. Epub 2011 Jul 13.
The enzymatically active region of amylopullulanase from Thermoanaerobacterium saccharolyticum NTOU1 (TsaNTOU1Apu) was identified by truncation mutagenesis. Two truncated TsaNTOU1Apu enzymes, TsaNTOU1ApuM957 and TsaNTOU1ApuK885, were selected and characterized. Both TsaNTOU1ApuM957 and TsaNTOU1ApuK885 showed similar specific activities toward various substrates. The overall catalytic efficiency (k (cat)/apparent K (m)) for the soluble starch or pullulan substrate, however, was 20-25% lower in TsaNTOU1ApuK885 than in TsaNTOU1ApuM957. Both truncated enzymes exhibited similar thermostability and substrate-binding ability against the raw starch. The fluorescence and circular dichroism spectrometry studies indicated that TsaNTOU1ApuK885 retained an active folding conformation similar to that of TsaNTOU1ApuM957. These results indicate that a large part of the TsaNTOU1Apu, such as the C-terminal carbohydrate-binding module family 20, the second fibronectin type III, and a portion of the first FnIII motifs, could be removed without causing a serious aberrant structural change or a dramatic decrease in hydrolysis of soluble starch and pullulan.
嗜热厌氧杆菌 NTOU1 （Thermoanaerobacterium saccharolyticum NTOU1）来源的支链淀粉酶的酶活性区域通过截断突变鉴定。选择并表征了两种截断的 TsaNTOU1Apu 酶，TsaNTOU1ApuM957 和 TsaNTOU1ApuK885。TsaNTOU1ApuM957 和 TsaNTOU1ApuK885 对各种底物均显示出相似的比活性。然而，TsaNTOU1ApuK885 对可溶性淀粉或普鲁兰底物的总催化效率（kcat/表观 Km）比 TsaNTOU1ApuM957 低 20-25%。两种截断的酶对生淀粉表现出相似的热稳定性和底物结合能力。荧光和圆二色性光谱研究表明，TsaNTOU1ApuK885 保留了与 TsaNTOU1ApuM957 相似的活性折叠构象。这些结果表明，TsaNTOU1Apu 的很大一部分，如 C 末端碳水化合物结合模块家族 20、第二个纤维连接蛋白型 III 和第一 FnIII 基序的一部分，可以被去除，而不会导致严重的异常结构变化或可溶性淀粉和普鲁兰水解的急剧下降。
